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Escherichia coli YafP protein modulates DNA damaging property of the nitroaromatic compounds.

Gutierrez A, Elez M, Clermont O, Denamur E, Matic I - Nucleic Acids Res. (2011)

Bottom Line: Using a murine septicaemia model, we showed that YafP activity reduced the bacterial fitness in the absence of PolIV.The YafP antimutator activity was independent of the PolIV activity.Given that YafP was annotated as a putative acetyltransferase, it could be that YafP participates in the metabolic transformation of genotoxic compounds, hence modulating the balance between their mutagenicity and cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Faculté de Médecine Paris Descartes, Inserm U1001, Université Paris Descartes, Paris, France.

ABSTRACT
Escherichia coli SOS functions constitute a multifaceted response to DNA damage. We undertook to study the role of yafP, a SOS gene with unknown function. yafP is part of an operon also containing the dinB gene coding for DNA Polymerase IV (PolIV). Our phylogenetic analysis showed that the gene content of this operon is variable but that the dinB and the yafP genes are conserved in the majority of E. coli natural isolates. Therefore, we studied if these proteins are functionally linked. Using a murine septicaemia model, we showed that YafP activity reduced the bacterial fitness in the absence of PolIV. Similarly, YafP increased cytotoxicity of two DNA damaging nitroaromatic compounds, 4-nitroquinoline-1-oxide (NQO) and nitrofurazone, in the absence of PolIV. The fact that PolIV counterbalances YafP-induced cytotoxicity could explain why these two genes are transcriptionally linked. We also studied the involvement of YafP in genotoxic-stress induced mutagenesis and found that PolIV and YafP reduced NQO-induced mutagenicity. The YafP antimutator activity was independent of the PolIV activity. Given that YafP was annotated as a putative acetyltransferase, it could be that YafP participates in the metabolic transformation of genotoxic compounds, hence modulating the balance between their mutagenicity and cytotoxicity.

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Fitness of CFT073 dinB yafP operon mutants. (A and B) Survival and fitness of dinB yafP operon mutants during in vitro competitions. (A) Competitive index of different mutants during batch culture competitions in LB: wild type (WT) and dinB (filled diamond); WT and dinB yafP (filled square); dinB yafP::FRT and dinB yafP::CmR (filled circle). The last competition was used as an evaluation of the cost of the CmR cassette. (B) Competitive index of different strains co-inoculated and incubated in LB for 5 days. Each point represents the mean (±SE) values from 3 independent experiments. (C and D) fitness of dinB yafP operon mutants during septicemia in the mouse model. (C) Competitive index of the WT and the different mutants, as well as measurements of CmR cassette cost during the competition. The latter was performed by competing yafP::FRT with yafP::CmR, and dinB yafP::FRT I with dinB yafP::CmR I mutants. (D) Competitive index of the WT and the dinB strain, which carried the pGB2 plasmid or the pGB2 bearing the functional dinB gene. Each point represents the mean (±SE) values from, at least 15 mice inoculated in at least three independent experiments. Asterisks show the competition for which the competitive index was significantly different (Wilcoxon test P < 0.05).
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Figure 3: Fitness of CFT073 dinB yafP operon mutants. (A and B) Survival and fitness of dinB yafP operon mutants during in vitro competitions. (A) Competitive index of different mutants during batch culture competitions in LB: wild type (WT) and dinB (filled diamond); WT and dinB yafP (filled square); dinB yafP::FRT and dinB yafP::CmR (filled circle). The last competition was used as an evaluation of the cost of the CmR cassette. (B) Competitive index of different strains co-inoculated and incubated in LB for 5 days. Each point represents the mean (±SE) values from 3 independent experiments. (C and D) fitness of dinB yafP operon mutants during septicemia in the mouse model. (C) Competitive index of the WT and the different mutants, as well as measurements of CmR cassette cost during the competition. The latter was performed by competing yafP::FRT with yafP::CmR, and dinB yafP::FRT I with dinB yafP::CmR I mutants. (D) Competitive index of the WT and the dinB strain, which carried the pGB2 plasmid or the pGB2 bearing the functional dinB gene. Each point represents the mean (±SE) values from, at least 15 mice inoculated in at least three independent experiments. Asterisks show the competition for which the competitive index was significantly different (Wilcoxon test P < 0.05).

Mentions: In order to find a phenotype associated with the YafP protein, we constructed a series of mutant derivatives of the CFT073 strain: dinB, yafP and dinB yafP. First, we assessed the growth rates of these mutants relative to the CFT073 strain in different media (LB, M9 minimal medium with different carbon sources) and found no differences. CmR cassette used for gene deletion has no effect on growth. Because competitions allow measurements of small differences in growth rates and survival, we performed in vitro batch culture competitions in LB between WT strain and dinB or dinB yafP mutants (Figure 3A). We found that there was no difference in fitness between these strains during five cycles, where each cycled allowed about 10 generations of growth. In addition, we monitored survival of different strains in mixed stationary phase cultures for 5 days (Figure 3B). There was no difference between the WT strain and dinB or dinB yafP mutants in this condition either. Hence, the phenotype of the yafP gene mutant cannot be detected under standard laboratory in vitro growth conditions.Figure 3.


Escherichia coli YafP protein modulates DNA damaging property of the nitroaromatic compounds.

Gutierrez A, Elez M, Clermont O, Denamur E, Matic I - Nucleic Acids Res. (2011)

Fitness of CFT073 dinB yafP operon mutants. (A and B) Survival and fitness of dinB yafP operon mutants during in vitro competitions. (A) Competitive index of different mutants during batch culture competitions in LB: wild type (WT) and dinB (filled diamond); WT and dinB yafP (filled square); dinB yafP::FRT and dinB yafP::CmR (filled circle). The last competition was used as an evaluation of the cost of the CmR cassette. (B) Competitive index of different strains co-inoculated and incubated in LB for 5 days. Each point represents the mean (±SE) values from 3 independent experiments. (C and D) fitness of dinB yafP operon mutants during septicemia in the mouse model. (C) Competitive index of the WT and the different mutants, as well as measurements of CmR cassette cost during the competition. The latter was performed by competing yafP::FRT with yafP::CmR, and dinB yafP::FRT I with dinB yafP::CmR I mutants. (D) Competitive index of the WT and the dinB strain, which carried the pGB2 plasmid or the pGB2 bearing the functional dinB gene. Each point represents the mean (±SE) values from, at least 15 mice inoculated in at least three independent experiments. Asterisks show the competition for which the competitive index was significantly different (Wilcoxon test P < 0.05).
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Related In: Results  -  Collection

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Figure 3: Fitness of CFT073 dinB yafP operon mutants. (A and B) Survival and fitness of dinB yafP operon mutants during in vitro competitions. (A) Competitive index of different mutants during batch culture competitions in LB: wild type (WT) and dinB (filled diamond); WT and dinB yafP (filled square); dinB yafP::FRT and dinB yafP::CmR (filled circle). The last competition was used as an evaluation of the cost of the CmR cassette. (B) Competitive index of different strains co-inoculated and incubated in LB for 5 days. Each point represents the mean (±SE) values from 3 independent experiments. (C and D) fitness of dinB yafP operon mutants during septicemia in the mouse model. (C) Competitive index of the WT and the different mutants, as well as measurements of CmR cassette cost during the competition. The latter was performed by competing yafP::FRT with yafP::CmR, and dinB yafP::FRT I with dinB yafP::CmR I mutants. (D) Competitive index of the WT and the dinB strain, which carried the pGB2 plasmid or the pGB2 bearing the functional dinB gene. Each point represents the mean (±SE) values from, at least 15 mice inoculated in at least three independent experiments. Asterisks show the competition for which the competitive index was significantly different (Wilcoxon test P < 0.05).
Mentions: In order to find a phenotype associated with the YafP protein, we constructed a series of mutant derivatives of the CFT073 strain: dinB, yafP and dinB yafP. First, we assessed the growth rates of these mutants relative to the CFT073 strain in different media (LB, M9 minimal medium with different carbon sources) and found no differences. CmR cassette used for gene deletion has no effect on growth. Because competitions allow measurements of small differences in growth rates and survival, we performed in vitro batch culture competitions in LB between WT strain and dinB or dinB yafP mutants (Figure 3A). We found that there was no difference in fitness between these strains during five cycles, where each cycled allowed about 10 generations of growth. In addition, we monitored survival of different strains in mixed stationary phase cultures for 5 days (Figure 3B). There was no difference between the WT strain and dinB or dinB yafP mutants in this condition either. Hence, the phenotype of the yafP gene mutant cannot be detected under standard laboratory in vitro growth conditions.Figure 3.

Bottom Line: Using a murine septicaemia model, we showed that YafP activity reduced the bacterial fitness in the absence of PolIV.The YafP antimutator activity was independent of the PolIV activity.Given that YafP was annotated as a putative acetyltransferase, it could be that YafP participates in the metabolic transformation of genotoxic compounds, hence modulating the balance between their mutagenicity and cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Faculté de Médecine Paris Descartes, Inserm U1001, Université Paris Descartes, Paris, France.

ABSTRACT
Escherichia coli SOS functions constitute a multifaceted response to DNA damage. We undertook to study the role of yafP, a SOS gene with unknown function. yafP is part of an operon also containing the dinB gene coding for DNA Polymerase IV (PolIV). Our phylogenetic analysis showed that the gene content of this operon is variable but that the dinB and the yafP genes are conserved in the majority of E. coli natural isolates. Therefore, we studied if these proteins are functionally linked. Using a murine septicaemia model, we showed that YafP activity reduced the bacterial fitness in the absence of PolIV. Similarly, YafP increased cytotoxicity of two DNA damaging nitroaromatic compounds, 4-nitroquinoline-1-oxide (NQO) and nitrofurazone, in the absence of PolIV. The fact that PolIV counterbalances YafP-induced cytotoxicity could explain why these two genes are transcriptionally linked. We also studied the involvement of YafP in genotoxic-stress induced mutagenesis and found that PolIV and YafP reduced NQO-induced mutagenicity. The YafP antimutator activity was independent of the PolIV activity. Given that YafP was annotated as a putative acetyltransferase, it could be that YafP participates in the metabolic transformation of genotoxic compounds, hence modulating the balance between their mutagenicity and cytotoxicity.

Show MeSH
Related in: MedlinePlus